Purpose:
Age-related maculopathy susceptibility 2 (ARMS2) is a small (11 kDa), primate-specific protein. Variants in the chromosomal region harboring the ARMS2 gene are strongly associated with an elevated risk for age-related macular degeneration (AMD), a leading cause of legal blindness in the developed word. In spite of intensive research, the biological function of ARMS2 remains elusive. Our previous work has indicated that ARMS2 is a genuine secreted protein, and as such a constituent of the extracellular matrix. Strikingly, ARMS2 lacks any predictable targeting signal and the mechanism responsible for its secretion has remained elusive. In this study, we sought to identify contributions of this novel secretion pathway.

Methods:
Immortalized human retinal epithelial cells (ARPE-19) were transfected by plasmids coding for ARMS2. Production and intracellular localization of the protein were examined by immunocytochemistry. Morphological analysis has been performed by co-staining for well-established markers of the canonical secretory pathway. Mutations of the original ARMS2 sequence carrying the desired substitutions were constructed by cloning. Cellular compartments were isolated by differential solubilization and tested for the presence of ARMS2 using Western blotting. Quantitative proteomic approaches (stable isotope labeling with amino acids in cell culture, SILAC) were used to identify proteins involved in the non-canonical secretion of ARMS2.

Results:
Immunocytochemical analyses and the Western blotting experiments indicate that ARMS2 accumulates in Calnexin-positive (ER-derived) vesicular compartments, but absent from other domains like tubular ER or Golgi. In line with this, the disruption of the Golgi apparatus does not influence ARMS2 secretion. Mutation analysis of the ARMS2 sequence identified three regions, which are indispensable for protein export. Quantitative co-immunoprecipitation experiments delineate a unique set of proteins driving this non-canonical secretion.

Conclusions:
These data unveil a novel, Golgi-independent vesicular secretion pathway and a mechanism by which ARMS2 is transported to the extracellular space. Although the physiological function of ARMS2 still needs to be clarified, understanding its intracellular trafficking might provide knowledge for a future therapy caused by compromised extracellular matrix in the back of the eye.